Cyanoacrylate tissue adhesives

ABSTRACT

A tissue adhesive including cyanoacrylate and polyethylene glycol (PEG), wherein the PEG remains a polyether and wherein the tissue adhesive is bioabsorbable. A tissue adhesive including cyanoacrylate bulk monomer and cyanoacrylate nanoparticles containing a therapeutic therein. A kit for applying tissue adhesive, including the tissue adhesive described above and an applicator. Methods of accelerating degradation of a cyanoacrylate tissue adhesive applying a tissue adhesive, closing an internal wound, administering a therapeutic to tissue, treating cancer, and preventing infection in a wound. Methods of method of making a bioabsorbable tissue adhesive and making a therapeutic tissue adhesive.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/742,868, which was filed on May 13, 2010 as the U.S. National Phaseof PCT/US07/84607, which was filed on Nov. 14, 2007, the contents ofeach are incorporated by reference herein in their entirety and for allpurposes.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to tissue treatments and sealants. Inparticular, the present invention relates to cyanoacrylate tissueadhesives.

(2) Description of Related Art

Several different techniques have been used for internal wound closureand tissue fixation such as sutures, tapes, staples, ligating clips, andadhesives. Adhesives are easy to apply and reduce physical pain as wellas the patient's anxiety associated with suture needles.

For internal applications, absorbable materials are highly preferred.Absorption eliminates the foreign body response characteristic of allpermanent synthetic implants, and eliminates the need for a secondsurgery for material removal. The most widely used absorbable materialsfor internal tissue fixation and wound closure are sutures formed ofdegradable poly alpha-hydroxy acids, for example, polyglactin 910(VICRYL®, Johnson and Johnson). Adhesives are particularly attractivebecause of their ability for simple, rapid application and curing.

Alkyl cyanoacrylate monomers have been used as tissue adhesives forseveral decades. Cyanoacrylate tissue adhesives are liquid monomers andcan polymerize quickly on contact with tissue surface creating a thin,flexible film. This polymer film creates a mechanical barrier, whichmaintains a natural healing environment. Short alkyl chaincyanoacrylates such as methyl, ethyl, and isopropylcyanoacrylates wereused as tissue adhesives, but because of their rapid in vivodegradation, resulting in significant tissue toxicity and inflammation,they were replaced by longer-chain cyanoacrylates such as butyl andoctylcyanoacrylate. Octylcyanoacrylate has received FDA approval in 1998and is marketed for skin wound closure after lacerations or incisions.

Cyanoacrylates offer a number of advantages, specifically as internaladhesives. Because their polymerization is initiated by functionalgroups found on biomolecules in tissue, their tissue bonding capabilityis greater than other types of adhesives. Cyanoacrylate adhesives alsoexhibit excellent bond strength and very rapid cure time. Cyanoacrylateesters with very short alkyl side chains (methyl and ethyl) are rapidlyabsorbed, but result in significant tissue toxicity. Cyanoacylate esterswith longer alkyl side chains exhibit reduced toxicity, but undergoextremely slow absorption.

In order to address this limitation, several methods have been developedto accelerate degradation of cyanoacrylate adhesives. One methodinvolves the introduction of a second ester bond during synthesis, whichincreases the susceptibility to hydrolysis (U.S. Pat. No. 6,620,846 toJonn, et al.). Another method pioneered by Shalaby in U.S. Pat. Nos.6,299,631 and 6,669,940 is the use of alkoxycyanoacrylate esters,specifically 2-methoxypropylcyanoacrylate, in combination withplasticizers of low molecular weight liquid polyesters, specificallythose based on oxalic acid. Several different polyester plasticizers canbe used, such as a copolyester comprising a polyethylene glycol (PEG)linked to succinate and oxalate units. Thus, the PEG is incorporated bychemical bonds into a polyester by copolymerization.

Recently, cyanoacrylate has been receiving much attention as a drug andgene delivery carrier, and anticancer drug loaded cyanoacrylatenanoparticles have been developed to treat cancer. Most drugs are eitherphysically or chemically immiscible with cyanoacrylates. In the case ofhydrophilic drugs, they cannot be miscible physically, and in the caseof hydrophobic drugs, they can be miscible, but drugs which containamine groups readily initiate cyanoacrylate polymerization. It would bedesirable to deliver therapeutics to the site to which the adhesive isapplied, such as antibiotics to prevent infection of a surgical site oranticancer agents to prevent cancer recurrence after surgery.

U.S. Pat. No. 6,746,667 to Badejo, et al., U.S. Pat. No. 6,942,875 toHedgpeth, and U.S. Pat. No. 6,767,552 to Narang (all assigned to ClosureMedical Co.), disclose haloprogin, an antifungal agent, mixed with2-octylcyanoacrylate (OCA) monomer by adding haloprogin directly intoOCA; and used said agent for different topical applications to remedyinfections such as tenia pedis, oral fungal, and skin yeast. Directmixing of haloprogin with the cyanoacrylate is possible becausehaloprogin is hydrophobic and does not contain any amine group in thestructure that would initiate cyanoacrylate polymerization. Thus, such asystem would not work with drugs that are hydrophilic or which haveamine groups in their structure.

U.S. Pat. No. 6,086,906 to Greff, et al. (assigned to Medlogic GlobalCo.) discloses various antimicrobial agents mixed directly withoctylcyanoacrylate, but only polyvinylpyrrolidone iodine complex wascompatible with the cyanoacrylate and showed a broad spectrum ofantibacterial activity. Polyvinylpyrrolidone is a polymer and has atertiary amine group in its structure, but it is used make a complexwith iodine. This tertiary amine cannot initiate cyanoacrylatepolymerization.

U.S. Pat. No. 6,974,585 to Askill discloses a cyanoacrylate formulationcontaining mixed antibiotics (0.3% neomycin, 0.15% polymyxin B, and0.55% bacitracin zinc). These drugs, however, form a suspension thatdoes not mix with cyanoacrylate monomer homogeneously and they form asuspension. The formulation must be prepared in the operating room on anas needed basis and used immediately; which is typically not convenient,especially for consumer use.

Therefore, there is a need for a cyanoacrylate adhesive (1) that isreadily absorbable in the body; (2) that does not cause significanttissue toxicity; (3) that can transport drug molecules effectively at asite of application for therapeutic treatment; and (4) that is aformulation that is easy to use.

BRIEF SUMMARY OF THE INVENTION

The present invention provides for tissue adhesives includingcyanoacrylate and polyethylene glycol (PEG), wherein the PEG remains apolyether and wherein the tissue adhesive is bioabsorbable. The presentinvention provides for tissue adhesives including cyanoacrylate bulkmonomer and cyanoacrylate nanoparticles containing a therapeutictherein.

The present invention further provides kits for applying tissueadhesives, including the above-described tissue adhesives and anapplicator. A method is provided for applying tissue adhesives totissue.

A method of closing an internal wound is provided by applying a tissueadhesive to a wound site, wherein the tissue adhesive includescyanoacrylate and PEG and is bioabsorbable, allowing the wound to heal,and degrading the tissue adhesive by accelerated hydrolytic degradationdue to water uptake by the PEG.

A method of sealing tissue internally is provided by applying tissueadhesive including cyanoacrylate and PEG, wherein the tissue adhesive isbioabsorbable to tissue, and sealing the tissue.

Methods are further provided of (1) administering therapeutics totissue; (2) treating cancer; and (3) preventing infection in a wound;all of which can be performed by applying a tissue adhesive includingcyanoacrylate bulk monomer and cyanoacrylate nanoparticles containing atherapeutic, and releasing the therapeutic from the cyanoacrylatenanoparticles to the tissue.

A method is provided of accelerating degradation of a cyanoacrylatetissue adhesive by adding PEG to cyanoacrylate to create a cyanoacrylatetissue adhesive, applying the cyanoacrylate tissue adhesive to tissue,and degrading the tissue adhesive by accelerated hydrolytic degradationdue to water uptake by the PEG and rapidly clearing the PEG through apatient's kidneys.

Methods are also provided for making a bioabsorbable tissue adhesive andfor making a therapeutic tissue adhesive.

BRIEF DESCRIPTION ON THE DRAWINGS

Other advantages of the present invention will be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings, wherein:

FIG. 1 is a bar graph showing a degradation study of variouscyanoacrylate adhesive formulations;

FIG. 2 is a bar graph showing a bond strength test of cyanoacrylatederivatives using rat skin;

FIGS. 3 A-3F are photographs of explants of various cyanoacrylateadhesives at 7 days post-surgery;

FIGS. 4A-4H are photographs of explants of various cyanoacrylateadhesives at 4 weeks post-surgery;

FIGS. 5A-5H are photographs of explants of various cyanoacrylateadhesives at 12 weeks post-surgery;

FIGS. 6A-6P are fluorescent microscopy images evaluating cellularinfiltration of cyanoacrylate implants at 7 days post-surgery byimmunohistological staining, red: Macrophage (Mc) and Fibroblast (Fb)positive cells, blue: DAPI stained non-specific cells, green:cyanoacrylate adhesive;

FIGS. 7A-7P are fluorescent microscopy images evaluating cellularinfiltration of cyanoacrylate implants at 4 weeks post-surgery byimmunohistological staining, red: Macrophage (Mc) and Fibroblast (Fb)positive cells, blue: DAPI stained non-specific cells, green:cyanoacrylate adhesive;

FIGS. 8A-8P are fluorescent microscopy images evaluating cellularinfiltration of cyanoacrylate implants at 12 weeks post-surgery byimmunohistological staining, red: Macrophage (Mc) and Fibroblast (Fb)positive cells, blue: DAPI stained non-specific cells, green:cyanoacrylate adhesive;

FIG. 9 is a bar graph showing in vitro cytotoxicity of cyanoacrylatederivatives using Human dermal fibroblasts;

FIGS. 10A (OCA+MCA), 10B (OCA+MC A/PEG), 10E (MCA), and 10F (MCA/PEG)are phase contrast images showing the interface between thecyanoacrylate adhesives and the surrounding collagen films. FIGS. 10C(OCA+MCA), 10D (OCA+MCA/PEG), 10G (MCA), and 10H (MCA/PEG) arecorresponding fluorescent images showing the location of adherent cells;

FIG. 11 is a flow chart showing the preparation of therapeutic-loadedcyanoacrylate tissue adhesives;

FIG. 12 is an ^(X)H-NMR spectrum of 5-FU-loaded polymyxin B-loadedpolybutylcyanoacrylate (PBCA) particles; and

FIGS. 13A-13D are photographs showing an antibacterial activity test ofpolymyxin B-loaded (PBCA) paticles by the agar diffusion method, 13Ashows 10 mg polymyxin B, 13B shows 10 mg PBCA, 13C shows 10 mg polyB/PBCA-1 (equivalent to 1.93 mg of free polymyxin B), and 13D shows 10mg poly B/PBCA-2 (equivalent to 1.24 mg of free polymyxin B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel cyanoacrylate tissue adhesives. Ina first embodiment of the invention, a bioabsorbable cyanoacrylatetissue adhesive is formed from a composition including cyanoacrylate andpolyethylene glycol (PEG). A second embodiment of the invention furtherprovides a composition including cyanoacrylate bulk monomer andcyanoacrylate nanoparticles containing an effective amount of atherapeutic therein.

The novel bioabsorbable cyanoacrylate tissue adhesive formulation is ablend of cyanoacrylate (also referred to herein as CA) and a lowmolecular weight polyether. The polyether can be any low molecularweight homopolymer such as polyethylene glycol (PEG) or various di- andtri-block copolymers of polyethylene glycol and polypropylene glycol,such as, but not limited to, PLURONIC® (BASF). Most preferably, thepolyether is PEG.

The bioabsorbable cyanoacrylate adhesives described herein are shown tohave an accelerated degradation rate compared to that of an adhesive ofa pure cyanoacrylate composition. Unlike compositions described above inthe prior art, the present invention is not a copolymer of cyanoacrylateand PEG, but rather the PEG remains a polyether. Polyethers, includingPEG, are essentially non-degradable in the human body. However, they arehighly water-soluble and rapidly cleared through the kidneys. Because ofits hydrophilic and water soluble properties, PEG acceleratescyanoacrylate degradation by increasing water uptake into the curedadhesive, accelerating hydrolytic degradation. PEG is also known to actas a plasticizer, increasing the viscosity of the adhesive.

Two potentially useful formulations have been developed as preferredembodiments. The first formulation, MCA/PEG, is a blend ofmethoxyisopropylcyanoacrylate (MCA) with a low molecular weight PEG(MW=600) at 85:15 (v/v % MCA/PEG) ratio. A thin film of MCA/PEG appliedto an in viva incision created in the rat hind limb muscle isessentially degraded in 12 weeks, and degraded more extensively than MCAalone. This is the most rapidly degrading formulation developed.OCA+MCA/PEG is a second potentially useful formulation, consisting of a50:50 v/v % mixture of octylcyanoacrylate (OCA) and MCA formulated withPEG at a 85:15 (v/v % CA/PEG) ratio. While exhibiting slower degradationthan MCA/PEG, this formulation shows accelerated degradation relative toa composition of OCA+MCA (50:50 v/v %) without PEG. This formulationalso exhibits significantly increased tissue bond strength relative toMCA/PEG. While the addition of PEG weakens the bond strength, OCAfortifies the bond strength.

The bioabsorbable cyanoacrylate adhesives can be used in many differentapplications, such as, but not limited to, cardiac surgery, generalwound closure, hernia surgery, artheroscopic surgery, endoscopicsurgery, and any type of major or minor surgery. Further, thebioabsorbable cyanoacrylate adhesive can be used as an internal sealant,for such applications as sealing tissue together, among others.

In other words, the present invention provides for a method of sealingtissue internally, including the steps of applying the tissue adhesiveof the first embodiment to tissue, and sealing the tissue. The presentinvention also provides for a method of closing an internal wound,including the steps of applying the tissue adhesive of claim of thefirst embodiment to a wound site, allowing the wound to heal, anddegrading the tissue adhesive by accelerated hydrolytic degradation dueto water uptake by the PEG.

In use, the bioabsorbable cyanoacrylate adhesive is applied to thedesired tissue area as a liquid which then polymerizes upon contact withtissue. The polymerized patch of bioabsorbable cyanoacrylate adhesiveallows the tissue to heal properly. Over time, water is drawn into theadhesive, causing it to degrade. The components of the adhesive then arecleared from the body.

Therefore, the present invention provides for a method of acceleratingdegradation of a cyanoacrylate tissue adhesive, including the steps ofadding PEG to cyanoacrylate to create a cyanoacrylate tissue adhesive,applying the cyanoacrylate tissue adhesive to tissue, and degrading thetissue adhesive by accelerated hydrolytic degradation due to wateruptake by the PEG and rapidly clearing the PEG through a patient'skidneys.

The second embodiment of the invention pertains to a method for creatingstable formulations of therapeutic-containing cyanoacrylate adhesives.Hereinafter, the therapeutic can also be referred to as a drug. Themethod involves the entrapment of the therapeutics inpolyalkylcyanoacrylate nanoparticles. Essentially, this process createsa stable coating of polymerized cyanoacrylate around the therapeuticmolecule/compound. Once purified and dried, the therapeutic-loadedcyanoacrylate nanoparticles can be homogeneously mixed with bulkcyanoacrylate monomer without initiating polymerization. Once the liquidadhesive is applied to tissue and polymerizes, the cyanoacrylatenanoparticles are degraded by hydrolysis of the cyanoacrylate ester,becoming water soluble cyanoacrylic acid and releasing free therapeuticat the surface of the wound or tissue. Dosing can be controlled by theamount of nanoparticles initially loaded. The choice of cyanoacrylatealkyl chain length and its effect on degradation rate can also be usedto control dosing.

Complex mixtures of multiple therapeutics such as those used incommercial antibiotic ointments (e.g. polysporin, which containspolymyxin B, Bacitracin, and Gramacidin as antibiotics and lidocaine aslocal anesthetics) can be readily created by mixing multiple types oftherapeutic-loaded nanoparticles with the adhesive. Primary applicationsinclude antibiotic delivery for reduction of wound infection anddelivery of anticancer chemotherapeutic drugs to wound sites followingremoval of malignant tissue. Other applications include nicotine orglucose delivery in a patch of the polymerized cyanoacrylate adhesive.In other words, a patch can be made out of the cyanoacrylate liquidadhesive that contains a therapeutic, and the patch can be applied to adesired area of tissue. When the patch is applied to the tissue, it isdegraded as described above and releases the therapeutic. Such patchescan be controlled-release according to the methods of controlling dosingdescribed above. Table I below shows various therapeutics that can beloaded in CA nanoparticles for topical application. The therapeuticslisted therein are not exhaustive, and any suitable therapeutic can beloaded into CA nanoparticles for delivery through the cyanoacrylateadhesive.

TABLE I Drugs for use in topical application Antifungal AnticancerAntiinflammatory Antibacterial agents agents agents drugs Polymyxin Bsulfate Terb marine 5-Fluorouracil Ibuprofen Neomycin sulfateClotrimazole Doxorubicin Ketoprofen Gentamycin sulfate KetokonazolePaclitaxel Dexamethas one Gramicidin Nystatin Predisolone ZincBacitracin Amphotericin B Hydroxyquinolone

For example, the nanoparticles can contain an anticancer agent. Thus,the present invention provides for a method of treating cancer,including the steps of applying a tissue adhesive includingcyanoacrylate bulk monomer and cyanoacrylate nanoparticles containing aneffective amount of an anticancer agent, releasing the anticancer agentfrom the cyanoacrylate nanoparticles to the tissue, and treating cancer.

The nanoparticles can also contain an antibiotic. Thus, the presentinvention provides for a method of preventing infection in a wound,including the steps of applying a tissue adhesive includingcyanoacrylate bulk monomer and cyanoacrylate nanoparticles containing aneffective amount of an antibiotic therapeutic to a wound, releasing theantibiotic therapeutic from the cyanoacrylate nanoparticles to thetissue, and preventing infection in the wound.

The second embodiment with therapeutic-loaded CA nanoparticles can bepracticed in combination with the first embodiment, i.e., abioabsorbable CA and PEG bulk monomer. Thus, the therapeutic-loadednanoparticles can effectively be released internally or at an outer sitewhere quick degradation of the CA adhesive is desired. This isespecially desirable where a therapeutic needs to be delivered to aninternal surgical site. Therapeutic release from nanoparticles incyanoacrylate adhesive can be substantially accelerated by theincorporation of PEG within the cyanoacrylate adhesive. Increased wateruptake mediated by PEG can accelerate nanoparticle degradation andtherapeutic release from nanoparticles, as well as increase thepermeability of the adhesive to the therapeutic, thus facilitating itsdiffusion from the adhesive into the surrounding tissue.

In use, the therapeutic-containing cyanoacrylate adhesive is applied asa liquid to the desired tissue area, which then polymerizes into a patchupon contact with the tissue. Alternatively, the therapeutic-containingcyanoacrylate adhesive is already in a patch form as described above andapplied as such to tissue. Over time, the cyanoacrylate nanoparticles ofthe patch release the therapeutic at the surface of the tissue as thenanoparticles degrade. Thus, not only does the tissue heal byapplication of the cyanoacrylate adhesive, but the therapeutic alsopromotes healing and/or prevention of further disease.

Several kits are provided for the present invention. A kit generallyincludes an applicator containing the cyanoacrylate bulk monomercomposition. A kit for the second embodiment of the invention furtherincludes the therapeutic-loaded nanoparticles. The therapeutic-loadednanoparticles can be directly mixed in with the cyanoacrylate bulkmonomer inside the applicator. A compartment containing thecyanoacrylate tissue adhesive inside the applicator can be activatedwhen needed, and the cyanoacrylate tissue adhesive flows out of theapplicator to be applied to tissue. A sponge can be included on the tipof the applicator for easy application.

It is also envisioned that the therapeutic-loaded nanoparticles can becontained in a compartment within the applicator, separate from thecyanoacrylate bulk monomer. For example, a sponge tip of the applicatorcan contain an antibiotic in a water-based solution or micelle and aseparate compartment can contain the cyanoacrylate bulk monomer. Whenthe compartment is opened or activated, the cyanoacrylate bulk monomerflows through the sponge tip and picks up the antibiotic before flowingto a surface of the sponge. Thus, when the sponge surface is applied toa wound, the adhesive contains both the bulk cyanoacrylate monomer andthe antibiotic. Another applicator can contain two compartments, onewith bulk cyanoacrylate monomer, and one with a therapeutic. Forexample, the compartments can be at opposite ends in a single tube, orside by side vertically. The compartment with the therapeutic can beopened or activated to apply the therapeutic first, and then thecompartment with the bulk cyanoacrylate monomer can be opened to applythe adhesive.

Individual applicators can be packaged separately to maintain sterileconditions. For example, each applicator can be packaged in plastic orany other suitable enclosing material. Multiple applicators can then bepackaged in a box for shipping.

The compounds of the present invention are administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In the method of the present invention, the compounds of the presentinvention can be administered in various ways. It should be noted thatthey can be administered as the compound and can be administered aloneor as an active ingredient in combination with pharmaceuticallyacceptable carriers, diluents, adjuvants and vehicles. The compounds canbe administered in any suitable way. The patients being treated arewarm-blooded animals and, in particular, mammals including human beings.The pharmaceutically acceptable carriers, diluents, adjuvants andvehicles as well as implant carriers generally refer to inert, nontoxicsolid or liquid fillers, diluents or encapsulating material not reactingwith the active ingredients of the invention.

The doses can be single doses or multiple doses over a period of severaldays. The treatment generally has a length proportional to the length ofthe disease process and drug effectiveness and the patient species beingtreated.

When administering the compound of the present invention with the CAnanoparticles containing therapeutics parenterally, it will generally beformulated in a unit dosage injectable form (solution, suspension,emulsion). The pharmaceutical formulations suitable for injectioninclude sterile aqueous solutions or dispersions and sterile powders forreconstitution into sterile injectable solutions or dispersions. Thecarrier can be a solvent or dispersing medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), suitable mixtures thereof, andvegetable oils.

Proper fluidity of the therapeutics can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil,olive oil, soybean oil, corn oil, sunflower oil, or peanut oil andesters, such as isopropyl myristate, may also be used as solvent systemsfor compound compositions. Additionally, various additives which enhancethe stability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.According to the present invention, however, any vehicle, diluent, oradditive used would have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various of the other ingredients,as desired.

A pharmacological formulation of the therapeutics contained in the CAnanoparticles of the present invention can be administered to thepatient in an injectable formulation containing any compatible carrier,such as various vehicles, adjuvants, additives, and diluents; or thecompounds utilized in the present invention can be administeredparenterally to the patient in the form of slow-release subcutaneousimplants or targeted delivery systems such as monoclonal antibodies,vectored delivery, iontophoretic, polymer matrices, liposomes, andmicrospheres. Examples of delivery systems useful in the presentinvention include those disclosed in U.S. Pat. Nos. 5,225,182;5,169,383; 5,167,616; 4,959,217; 4,925, 678; 4,487,603; 4,486,194;4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other suchimplants, delivery systems, and modules are well known to those skilledin the art.

While specific embodiments are disclosed herein, they are not exhaustiveand can include other suitable designs and systems that vary in designs,methodologies, and transduction systems (i.e., assays) known to those ofskill in the art. In other words, the examples are provided for thepurpose of illustration only, and are not intended to be limiting unlessotherwise specified. Thus, the invention should in no way be construedas being limited to the following examples, but rather, should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

Example 1 Experimental Methods and Results

A series of experiments have been performed to evaluate the efficacy ofcyanoacrylate adhesive formulations incorporating PEG. Throughout allexperiments, PEG (MW=600) was added to cyanoacrylates at a 85:15 (v/vCyanoacrylate/PEG) ratio. Three cyanoacrylate compositions were used:octylcyanoacrylate (OCA), a 50:50 mixture of octylcyanoacrylate andmethoxyisopropylcyanoacrylate (OCA:MCA) andmethoxyisopropylcyanoacrylate (MCA). Pure cyanoacrylate formulationswere used throughout as controls for comparison to experimentalformulations containing PEG.

1. In Vitro Degradation Study

In vitro accelerated degradation studies have been performed to evaluatethe degradation rate of various cyanoacrylate adhesives formulated withPEG and without PEG. 20 μl of cyanoacrylate (CA) derivatives and CAderivatives/PEG (MW=600) formulations were spread as a thin layer on theglass microscope slides and allowed to cure overnight at roomtemperature. The resulting thin films (1.5 cm×1.5 cm) were weighed (W₀)and incubated in 10 ml 0.05N NaOH solution at 37° C. The films werecollected at 3, 7, 14, and 21 days and washed with distilled water twotimes. The washed films were freeze-dried and weighed (W₁) (n=3).Percent degradation was then calculated from the following formula:

% Degradation of cyanoacrylate derivatives=(W _(o) −W_(t))/W_(o)×100  (1)

FIG. 1 shows % degradation of various cyanoacrylate derivatives in 0.05N NaOH solution. Formulations based on cyanoacrylate monomers with shortalkyl side chains (MCA) exhibited significantly increased degradationrelative to those based on monomers with long alkyl side chains (OCA),consistent with previous reports in the literature. Formulationscomprising mixture of OCA:MCA (50:50) exhibited intermediate levels ofdegradation relative to each monomer. The addition of 15% PEGsignificantly increased in vitro degradation rate of all cyanoacrylateformulations.

2. Tissue Bond Strength Study

Rat skin samples were harvested from the backs of Sprague-Dawley ratsand cut into strips (1 cm in width, 5 cm in length). The strips were cutin half and 2 μl of various cyanoacrylate formulations were applied toeach side of cut edges to glue them. The glued strips were incubatedovernight at 4° C. in humidified chamber to allow them to cure. Bondstrength of samples (n=4) was tested using MTS Synergie 100 (MTS SystemCorporation). FIG. 2 shows the bond strength of various cyanoacrylateformulations. The addition of PEG significantly decreased tissue bondstrength relative to each corresponding formulation of cyanoacrylate.The MCA/PEG formulation exhibited significantly reduced tissue bondstrength relative to OCA and OCA/MCA formulations including PEG.

3. Immunohistochemical (IHC) Staining for Evaluation of In VivoBiocompatibility

Small surgical incisions (0.2 cm in depth, 1 cm in length) were made inboth hind limb muscles of adult Sprague-Dawley rats (n=4). One incisionwas closed by 1 drop of various cyanoacrylate formulations dispensedthrough a surgical syringe needle (25 G) and the other was closed byabsorbable suture (VICRYL, Ethicon) with and without injection ofequivalent volumes of PEG included in cyanoacrylate formulations ascontrols. In order to ensure proper identification of the injury sites,a single non-absorb-able suture was placed adjacent to each repairedincision site.

At 7 days, 4 weeks, and 12 weeks post-surgery, tissue explantscontaining the repaired incision sites and surrounding muscle tissuewere retrieved. The explants were photographed to document themacroscopic tissue response to the various cyanoacrylate formulationsand suture controls (FIGS. 3A-3H (7 days), 4A-4H (4 weeks), and 5A-5H(12 weeks)). Identification of specific cells types associated withwound healing and inflammation was performed by histological sectioningand immunohistochemical (IHC) staining. Tissue explants were embedded inOptimal cutting temperature (OCT) compound and sectioned perpendicularto the surface of the repaired muscle tissue. Sections (0.6 um thick)were transferred to glass microscope slides, fixed with methanol,permeabilized with 0.025% Triton in PBS solution, and stained withprimary antibodies directed against vimentin (fibroblasts) and ratmacrophages, and Alexa-594 conjugated secondary antibodies. DAPImounting media was used to counterstain all cellular nuclei. Sampleswere imaged by fluorescence microscopy (FIGS. 6A-6P, 7A-7P, 8A-8P).Specific cell types are identified in red, sutures and variouscyanoacrylate adhesives are shown in green, and cellular nuclei areshown in blue. Tables II, III, and IV below summarize macroscopic andhistological observations of the biocompatibility and degradation ofvarious cyanoacrylate formulations at 7 days, 4 weeks, and 12 weekspost-surgery, respectively.

TABLE II Evaluation of biocompatibility of various cyanoacrylateadhesives at 7 days post-surgery Degree of Vessel Degree of No. of No.of Sample Rat No. inflammation formation degradation macrophagesfibroblasts Suture 1 no no no ** ** 2 no no no ** ** 3 * no no ** ** 4no no no ** ** Sut/PEG 1 no no no ** ** 2 no no no *** ** 3 no no no **** 4 no no no *** ** OCA 1 no no no ** *** 2 no no no *** **** 3 * no no**** *** 4 ** ** no ***** *** OCA/PEG 1 * * no * *** 2 * * no *** ***3 * * no *** *** 4 * no no **** *** OCA + MCA 1 no no no * * 2 no nono * * 3 no no no * * 4 no no no ** ** OCA + MCA/ 1 no * no *** ** PEG 2no no no ** * 3 no no no ** * 4 no no no ** * MCA 1 no no no * * 2 no nono ** * 3 no no no *** * 4 no no no *** ** MCA/PEG 1 no no no * * 2 nono no ** * 3 no no no ** * 4 * no no *** ****

TABLE III Evaluation of biocompatibility of various cyanoacrylateadhesives at 4 weeks post-surgery Degree of Vessel Degree of No. of No.of Sample Rat No. inflammation formation degradation macrophagesfibroblasts Suture 1: Dead 2 no no no ** ** 3 no no no ** ** 4 no no no** ** Sut/PEG 1 no no no ** ** 2 no no no ** ** 3 no no no ** ** 4 no nono ** ** OCA 1 * no no *** **** 2 ** * no *** **** 3 * no no *** *** 4** * no **** **** OCA/PEG 1 *** no *** **** 2 *** no *** **** 3 * no no** ** 4 *** no *** **** OCA + 1: Dead * MCA 2 no no * ** * 3 no no *** * 4 no no * * * OCA + 1 no no ** *** ** MCA/PEG 2 no no ** ** * 3 nono ** ** * 4 no no ** ** * MCA 1 no no ** ** ** 2 no no ** * * 3 no no** ** * 4 no no ** * * MCA/PEG 1 no no *** ** * 2 no no *** * * 3 no no*** * * 4 no no *** ** *

TABLE IV Evaluation of biocompatibility of various cyanoacrylateadhesives at 4 weeks post-surgery Degree of Vessel Degree of No. of No.of Sample Rat. No. inflammation formation degradation macrophagesfibroblasts Suture 1: Dead 2 no no Degraded ** ** 3: Dead 4 no noDegraded * ** Sut/PEG 1 no no * ** 2 no no Degraded 0 ** 3 no no * * 4no no Degraded 0 ** OCA 1 no no * ** **** 2 no no * *** *** 3 * * * * *4 no no * * ** OCA/PEG 1 ** * * *** **** 2 * * * *** *** 3 no no * ***** 4 no no * *** *** OCA + 1: Dead MCA 2 no no ** ** ** 3: Dead 4 no no** ** ** OCA + 1 no no *** * ** MCA/PEG 2 ** 3 no no *** ** ** 4 no no*** *** ** MCA 1 no no *** ** ** 2 no no *** * ** 3 no * *** 0 * 4 no no** 0 * MCA/PEG 1 no no ***** * * 2 no no ***** * * 3 no no ***** * * 4no no ***** * **At 7 days post-surgery, macroscopic analysis of tissue explants revealedmild inflammation and swelling in response to OCA (2 of 4 rats) andOCA/PEG (all 4 rats) formulations. Suture controls and formulationsbased on OCA/MCA and MCA both with and without PEG appeared wellintegrated with the surrounding tissue. Suture controls showed littlechange at 4 weeks and were substantially degraded by 12 weeks. OCA andOCA/PEG samples exhibited minimal degradation at 4 or 12 weeks. Samplesof OCA/MCA and MCA-based formulations exhibited progressive degradationat 4 and 12 weeks, with MCA formulations showing the greatest degree ofdegradation. Inclusion of PEG in OCA/MCA and MCA formulations resultedin increased degradation relative to pure cyanoacrylate formulations.These observations parallel the results of the in vitro degradationanalysis with the degree of degradation proceeding in the orderMCA/PEG>MCA OC A+MC A/PEG>OC A-1-MC A>OC Al PEG>OCA. These results werefurther confirmed by visualization of the various formulations in thehistological analysis described below.

Histological analysis demonstrated that all implant materials promotedthe accumulation of an increased number of cells relative to uninjuredmuscle tissue, which exhibits low cellularity. This cellular capsulevaried in thickness as a function of the implant material andformulation. In general, macrophages were concentrated at the surface ofall implants, surrounded by fibroblasts. OCA-based formulations wereencapsulated by substantially thicker cell layers as compared to MCA andOCA/MCA formulations at both 7 day and 4 week time points.

4. In Vitro Cytotoxicity of CA Derivatives on Human Dermal Fibroblast(HDF) Cells

2 μl of various CA formulations were applied on thin collagen films in12 well plates and allowed to polymerize completely overnight at roomtemperature. One ml of Dulbecco's Minimal Essential Medium (DMEM)including 10% Serum and 1% Penicillin/Streptomycin was added into eachwell and incubated at 37° C. in a humidified CO₂ incubator to simulateadhesive degradation. Media samples were collected at 2, 7, and 14 daysand these adhesive-conditioned media (ACM) were stored at −20° C. untiluse. Human dermal fibroblast (HDF) cells were seeded in new 12-wellculture plates at a density of 8×10⁴ cells/well and cultured for 24hours to allow cell attachment. The media was replaced with the storedACM and cultured for 2 days at 37° C. to evaluate the in vitrocytotoxicity of the various CA derivatives. Cell cytotoxicity by thedegraded CA adhesives in ACM was evaluated by MTT assay. The principleof this assay is the addition of a dye which is degraded by enzymespresent in living cells, resulting in the formation of a coloredproduct. The product concentration, which is generally directlyproportional to the number of viable cells, is assayed byspectrophotometry. FIG. 9 shows the % cell viability after culture withACM generated in the presence of various CA formulations. ACM from allCA derivatives was not cytotoxic except for the ACM from OCA in the 2days case. In 7 and 14 days, ACM from CA derivatives without PEG showedno cytotoxicity and ACM from CA derivatives with PEG showed cytotoxicityin order of MCA>OCA+MCA>OCA.

5. PEG Incorporation Reduces Cell Adhesion to Polymerized Cyanoacrylates

2 μl aliquots of various cyanoacrylate formulations with and without PEGwere applied to thin collagen films in 24 well plates and allowed tocure overnight at room temperature. Human dermal fibroblasts were seededon the collagen film/polymerized adhesive surfaces at 8×10⁴ cells/welland cultured for 48 hours. The cells were stained with fluoresceindiacetate and propidium iodide to visualize live (green) and dead (red)cells respectively. FIGS. 10A (OCA+MCA), 10B (OCA+MCA/PEG), 10E (MCA),and 10F (MCA/PEG) are phase contrast images (black and white) showingthe interface between the cyanoacrylate adhesives and the surroundingcollagen films and corresponding fluorescent images FIGS. 10C (OCA+MCA),10D (OCA+MCA/PEG), 10G (MCA), and 10H (MCA/PEG) showing the location ofadherent cells. Fibroblasts readily adhered in large numbers to both MCAand OCA+MCA adhesive formulations and the surrounding collagen film. Incontrast, the incorporation of PEG into MCA and OCA+MCA adhesivesdramatically reduced adhesion to the polymerized adhesive surface. Theseresults suggest that incorporation of PEG reduces the adhesivity of thepolymerized adhesive and can reduce the occurrence of undesired tissueadhesions formed between the adhesive and surrounding tissue whenapplied for internal closure applications.

Example 2 Preparation of Drug-Loaded Poly(butylcyanoacrylate) (PBCA)Particles A. Polymyxin B-Loaded PBCA Nanoparticles

Polymyxin B-loaded polybutylcyanoacrylate (PBCA) particles were preparedby an anionic polymerization method (FIG. 11). Various amounts ofpolymyxin B were dissolved in various acidic polymerization mediumcontaining different non-ionic surfactants and then various amount ofbutylcyanoacrylate were slowly added into the polymyxin B-containingpolymerization medium under vigorous stirring. The polymerization of themonomer was allowed for four hours at room temperature under vigorousstirring to complete polymerization. When the polymerization wascompleted, the milky suspension was neutralized by 1N NaOH and thenfreeze-dried in a lyophilizer. The dried nanoparticles were resuspendedin 10 ml distilled water and centrifuged at 15,000 rpm (Beckmancentrifuge, JA 17 rotor, Beckman, USA) for an hour at 4° C. and theprocess was repeated two times to completely remove free drugs andsurfactant. The nanoparticles were suspended in water, freeze-dried, andkept in a vacuum desiccator.

B. 5-Fluorouracil-Loaded PBCA Nanoparticles

5-FU-loaded PBCA particles were prepared by anionic polymerization. 100mg of 5-Fluorouracil (5-FU) was dissolved into 10 ml polymerizationmedium containing citric acid (0.2 w/v %) and dextran 60-90 kDa (0.8 w/v%) and then 200 mg butylcyanoacrylate monomer was slowly added into 5-FUcontaining polymerization medium under vigorous stirring. Polymerizationof the monomer was allowed for four hours at room temperature undervigorous stirring to complete polymerization. Particles were purified bywashing, centrifugation and freeze-dried as described above.

Determination of Drug-Loading Efficiency A. Polymyxin B

The amount of polymyxin B-loaded in the PBCA particles was determined byan HPLC method. HPLC was performed using a reverse phase CI8 column(Regis 10 cm×4.6 mm, 3 um) and employing a binary gradient formed from0.1% TFA in 75% acetonitrile in water (solution A) and 0.1% TFA in water(solution B) with the following binary gradient: at t=0 min, B=0%, att=20 min, B=25%, at t=27, B=85%, and at t=30 min, B=85% at flow rate of0.5 ml/min. The mobile phase was monitored by measuring UV absorbance at220 nm. Quantitation of polymyxin B was done using a calibration curveconstructed from the peak area versus the concentration of standardsolution of polymyxin B. The polymyxin B loading efficiency in PBCAnanoparticles was determined as follows:

% loading=(D= _(t=0) −D _(t=4))/D _(t=0)×100  (2)

wherein D_(t=0): drug concentration at time 0, and D_(t=4): drugconcentration after polymerization at 4 hours. Table V below shows thecharacteristics of polymyxin B-loaded PBCA nanoparticles prepared byvarious polymerization conditions.

TABLE V Preparation of various polymyxin B-loaded nanoparticles AmountAmount Anionic Amount of Drug Miscibility of Drug of BCA Polymerizationnanoparticle loading with OCA (mg) (pi) Medium (10 ml) Aggregation (mg)(%) monomer 100 100 1 w/v % Pluronic no 94.3 23.23 Not F68 (pH 1.5)miscible, polymerization 100 100 1 w/v % Pluronic no 54.3 15.19Miscible, no P105 (pH 1.5) polymerization 200 1 w/v % Pluronic no 137.317.24 P105 (pH 1.5) 84.7 47.87 125.1 28.79 100 100 0.5 w/v % no 69.323.65 Miscible no Pluronic polymerization F68 and P105 (pH1.5) 100 200no 88.3 20.43 153.6 19.26 50 100 no 18.3 8.79 50 200 no 14.6 17.49*Miscibiity of nanoparticles with OCA monomer was determined by mixing10 mg of nanoparticles with 100 μl of OCA monomer.

B. 5-Fluorouracil

The amount of 5-FU incorporated in nanoparticles was determined byUV/VIS spectrophotometer. After completion of polymerization, the milkysuspension was filtered by 0.2 um membrane filter and the drugconcentration in filtrate was calculated using a standard curveconstructed from the UV absorbance at 266 nm versus the concentration ofstandard solution of 5-FU. The 5-FU loading efficiency in PBCAnanoparticles was determined by the same equation (2) as above.Drug-loaded PBCA nanoparticles were analyzed by ¹H-NMR using DMSO-d6 todetermine the structure of drug and polymer in nanoparticles (FIG. 12).Table VI below shows the characteristics of 5-FU-loaded PBCAnanoparticles prepared in various polymerization conditions.

TABLE VI Anionic Amount Amount Polymerization Drug Miscibility of Drugof BCA Medium Amount of loading with OCA (mg) (Hi) (10 ml) Aggregationnanoparticle (%) monomer 100 200 0.2% Citric acid yes (pH 2.5) 0.2%Citric acid no 134 mg 14.55% Miscible, (pH 2.5) and no 0.8% Dextranpolymerization 60-90 kDa 14.22% *Miscibility of nanoparticies with OCAmonomer was determined by mixing 10 mg of nanoparticies with 100 μl ofOCA monomer.

Evaluation of Antibacterial Activity of Polymyxin B-Loaded PBCANanoparticles

Antibacterial activity of polymyxin B-loaded PBCA nanoparticles wasmeasured indirectly by the agar diffusion method. 30 ml of nutrient agarwas added into each of four culture plates (90 mm diameter, 15 mm high).After solidifying, a cylindrical hole (13 mm in diameter) was made inthe center of the plate and the contents were removed. 10 mg ofpolymyxin B, PBCA nanoparticles, and two polymyxin B-loaded PBCAnanoparticles (Poly BIPBCA-1 and 2) were added into a hole of eachplate, respectively and a few drops of molten agar were added to fix thesamples. The plate were inoculated with Escherichia coli and incubatedat 37° C. for 24 hours and the zone of inhibition was determined (FIGS.13A-13D). Polymyxin B-loaded PBCA particles showed similar antibacterialactivity (FIGS. 13C and 13D) with free polymyxin B (FIG. 13A), whereasdrug-unloaded PBCA particles showed no antibacterial activity (FIG.13B).

To evaluate the duration of antibacterial activity of each sample, thecontent of each hole was removed after 24 hours of incubation andtransferred to a hole of a freshly prepared plate, incubated again for24 hours and the zone of inhibition measured. This procedure wascontinuously repeated until the inhibition of growth was no longerobserved. The size of inhibition zone decreased on each successive testperiod as shown in Table VII below. Polymyxin B loaded-PBCA-1 and -2(equivalent to 1.93 mg and 1.24 mg of free polymyxin B, respectively)particles showed same duration of antibacterial activity with freepolymyxin B although the amount of polymyxin B in the nanoparticles usedwas less than about 5 and 8 times of the free polymyxin B.

TABLE VII Antibacterial activity of polymyxin B-loaded PBCA particlesusing Escherichia coli Inhibition zone in days: Diameter (mm)^(a)Sample^(b) 1 2 3 4 5 6 Polymyxin B 36 38.3 28 25.1 22 c PBCA cPolyB/PBCA-1 28 25.1 21.5 18.5 17.5 c Poly B/PBCA-2 30 23.5 23.5 16 15.4c ^(a)Data include the original size of the sample (13 mm in diameter).^(b)10 mg of free polymyxin B and 10 mg of PBCA, PolyB/PBCA-1 and 2equivalent to 1.93 mg and 1.24 mg of free Poly B) were used for the testc Inhibition of growth not observed.

Discussion of Results

Example 1 evaluated the efficacy of cyanoacrylate adhesive formulationsincorporating PEG. Longer alkyl side chains showed longer degradationrates as compared to shorter alkyl side chains. Also, the presence ofPEG increased degradation rates in all cyanoacrylate formulations, andalso increased the bond strength with skin. In vivo biocompatibilitystudies showed that longer alkyl side chains showed the leastdegradation and thicker cell layers, and addition of PEG increaseddegradation. Cell cytotoxicity was evaluated, and PEG was found toreduce undesirable cell adhesion to the adhesive surface.

Example 2 showed that drug particles can be successfully loaded intoPCBA nanoparticles and released from the nanoparticles. Using thefindings from Example 1 in conjunction with the findings of Example 2,cyanoacrylate adhesive formulations can be made including PCBAdrug-containing nanoparticles to have the degradation rate, bondstrength, and cell adhesion desired.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology, which has been used is intended tobe in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

1. A tissue adhesive composition, comprising an adhesive comprising 85 v% of a cyanoacrylate monomer selected from the group consisting ofmethoxyisopropylcyanoacrylate and a combination of octylcyanoacrylateand methoxyisopropylcyanoacrylate in a 50:50 v % ratio, and 15 v % ofpolyethylene glycol (PEG), wherein said PEG remains a polyether, andwherein said tissue adhesive is degraded within 12 weeks when applied toan incision.
 2. The tissue adhesive composition of claim 1, wherein theadhesive further comprises polymerized cyanoacrylate nanoparticlescontaining an effective amount of a therapeutic.
 3. The tissue adhesivecomposition of claim 2, wherein said therapeutic is selected from thegroup consisting of antibacterial agents, antifungal agents, anticanceragents, and anti-inflammatory drugs.
 4. The tissue adhesive compositionof claim 3, wherein said therapeutic is an antibacterial agent selectedfrom the group consisting of polymyxin B sulfate, neomycin sulfate,gentamycin sulfate, gramicidin, zinc bacitracin, and hydroxyquinolone.5. The tissue adhesive composition of claim 3, wherein said therapeuticis an antifungal agent selected from the group consisting ofterbinafine, clotrimazole, ketokonazole, nystatin, and amphotericin B.6. The tissue adhesive composition of claim 3, wherein said therapeuticis an anticancer agent selected from the group consisting of5-fluorouracil, doxorubicin, and paclitaxel.
 7. The tissue adhesivecomposition of claim 3, wherein said therapeutic is an anti-inflammatoryagent selected from the group consisting of ibuprofen, ketoprofen,dexamethasone, and predisolone.
 8. The tissue adhesive composition ofclaim 2, wherein said composition is formulated in a patch.
 9. Thetissue adhesive composition of claim 8, wherein said composition isformulated in a patch and said therapeutic is chosen from the groupconsisting of nicotine and glucose.